JNK is a ubiquitously expressed serine/threonine kinase belonging, together with ERK (extracellular-regulated kinase) and p38, to the family of mitogen-activated protein kinases (MAPKs). (Kyriakis J M, MAP kinases and the regulation of nuclear receptors, Sci. STKE 2000 (48):pe1; Whitmarsh A J, Davis R J, Signal transduction by MAP kinases: Regulation by phosphorylation-dependent switches, Sci. STKE 1999 (1):pe1; Schramek H, MAP kinases: from intracellular signals to physiology and disease, News Physiol. Sci. 2002; 17:62-7; Ichijo H, From receptors to stress-activated MAP kinases, Oncogene 1999, 18(45):6087-93). MAPKs are important mediators of signal transduction from the cell surface to the nucleus, using phosphorylation cascades to generate a coordinated response by a cell to an external stimulus by phosphorylation of selected intracellular proteins, including transcription factors. Additionally, JNK also phosphorylates non-nuclear proteins, for example, IRS-1, and Bcl-2 family members. (Davis R J, MAPKs: new JNK expands the group, Trends Biochem. Sci. 1994; 9(11):470-3; Seger R, Krebs E G, The MAPK signaling cascade, FASEB J. 1995; 9(9):726-35; Fanger G R, Gerwins P, Widmann C, Jarpe M B, Johnson G L, MEKKs, GCKs, MLKs, PAKs, TAKs, and tpls: upstream regulators of the c-Jun amino-terminal kinases? Curr. Opin. Genet. Dev. 1997; 7(1):67-74).
JNK was first identified in the early 1990s, and the term is derived from cJun N-terminal kinase; its best-known substrate is the transcription factor cJun. (Hibi M, Lin A, Smeal T, Minden A, Karin M, Identification of an oncoprotein-and UV-responsive protein kinase that binds and potentiates the c-Jun activation domain, Genes Dev. 1993; 7(11):2135-48; Dunn C, Wiltshire C, MacLaren A, Gillespie D A, Molecular mechanism and biological functions of c-Jun N-terminal kinase signalling via the c-Jun transcription factor, Cell Signal 2002; 14(7):585-93; Ip Y T, Davis R J, Signal transduction by the c-Jun N-terminal kinase (JNK)—from inflammation to development, Curr. Opin. Cell Biol. 1998; 10(2):205-19). JNK is also known as SAPKα, stress-activated protein kinase α, since JNK is activated upon exposure of cells to pro-inflammatory cytokines, growth factors, and environmental stress, such as UV-irradiation or heat shock. (Hibi M, Lin A, Smeal T, Minden A, Karin M, Identification of an oncoprotein-and UV-responsive protein kinase that binds and potentiates the c-Jun activation domain, Genes Dev. 1993; 7(11):2135-48; Ip Y T, Davis R J, Signal transduction by the c-Jun N-terminal kinase (JNK)—from inflammation to development, Curr. Opin. Cell Biol. 1998; 10(2):205-19; Derijard B, Hibi M, Wu I H, Barrett T, Su B, Deng T, Karin M, Davies R J, JNK1: a protein kinase stimulated by UV light and Ha-Ras that binds and phosphorylates the c-Jun activation domain, Cell 1994; 76(6):1025-37; Kyriakis J M, Banerjee P, Nikolakaki E, Dai T, Rubie E A, Ahmad M F, Avruch J, Woodgett J R, The stress-activated protein kinase subfamily of c-Jun kinases, Nature 1994; 369(6476):156-60).
Because of the variety of stress responses in which JNK is involved, a single pathway for JNK activation does not appear to exist. Many stimulants cause JNK activation through more than one mechanism. Depending on the cell type and state, JNK can be involved in processes as divergent as apoptosis (Kyriakis J M, Avruch J, Mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation, Physiol. Rev. 2001; 81(2):807-869; Xie X, Gu Y, Fox T, Coll J T, Fleming M A, Markland W, Caron P R, Wilson K P, Su M S, Crystal structure of JNK3: a kinase implicated in neuronal apoptosis, Structure 1998; 6(8):983-91), cell proliferation (Yang Y M, Bost F, Charbono W, Dean N, McKay R, Rhim J S, Depatie C, Mercola D, C-Jun NH(2)-terminal kinase mediates proliferation and tumor growth of human prostate carcinoma, Clin. Cancer Res. 2003; 9(1):391-401; Schwabe R F, Bradham C A, Uehara T, Hatano E, Bennett B L, Schoonhoven R, Brenner D A, c-Jun-N-terminal kinase drives cyclin D1 expression and proliferation during liver regeneration, Hepatology 2003; 37(4):824-32), and cell differentiation (Li B, Tournier C, Davis R J, Flavell R A, Regulation of IL-4 expression by the transcription factor JunB during T helper cell differentiation, EMBO J. 1999; 18(2):420-32; Yang D D, Conze D, Whitmarsh A J, Barrett T, Davis R J, Ricón M, Flavell R A, Differentiation of CD4+ T cells to Th1 cells requires MAP kinase JNK2, Immunity 1998; 9(4):575-85); hence, JNK is an essential regulator of physiological and pathological processes.
Scleroderma
Scleroderma is a rare disease with a stable incidence of approximately 19 cases per 1 million persons. The exact cause of scleroderma is unknown. Abnormalities involve autoimmunity and alteration of endothelial cell and fibroblast function. Systemic scleroderma usually begins with skin thickening, usually of the fingers, accompanied by Raynaud's phenomenon. Raynaud's disease typically precedes further manifestations of systemic scleroderma. Early in the disease the affected skin may be puffy and soft. The usual location of greatest skin thickening and hardening is the face, hands and fingers. Sclerodactyly is frequently present. Tendon friction rubs are often palpable on exam and can be painful. With more advanced disease, digital ulcers and auto-amputation may occur. Gastrointestinal dismotility is a feature, often manifested by heartburn, or by diarrhea with malabsorption or pseudo-obstruction. New onset hypertension or renal insufficiency are manifestations of the associated vascular injury. Heart failure or arrhythmia are also possible due to cardiac fibrosis. (Hachulla E, Launay D, Diagnosis and classification of systemic sclerosis, Clin Rev Allergy Immunol 2010; 40(2):78-83).
The major manifestations of scleroderma and in particular of systemic sclerosis are inappropriate excessive collagen synthesis and deposition, endothelial dysfunction, spasm, collapse and obliteration by fibrosis. In terms of diagnosis, an important clinical parameter is skin thickening proximal to the metacarpophalangeal joints. Raynaud's phenomenon is a frequent, almost universal component of scleroderma. It is diagnosed by color changes of the skin upon cold exposure. Ischemia and skin thickening are symptoms of Raynaud's disease.
UV Injury and Sunburn
The skin is one of the largest body organs and functions as one of its major interfaces with the environment, including solar radiation. Exposure to solar radiation has the beneficial effects of stimulating the cutaneous synthesis of vitamin D and providing radiant warmth. (McStay, C M Elahi E J, Sunburn, eMedicine—Online Medical Reference Textbook (last modified May 18, 2010) (online), (retrieved on 2010-08-30). Retrieved from the internet: <URL: http://emedicine.medscape.com/article/773203-overview>). Unfortunately, when the skin is subjected to excessive radiation in the ultraviolet range, deleterious effects, such as sunburn, occur. Sunburn is an acute cutaneous inflammatory reaction that follows excessive exposure of the skin to ultraviolet radiation (UVR). The inflammatory response occurs within 2-6 hours after exposure and peaks at 20-24 hours with symptoms such as erythema, warmth, tenderness, edema, and blistering (severe cases). Acute UVR injury will also lead to apoptosis of keratinocytes resulting in skin injury and skin remodeling. (Merryman, J. I., Neilsen, N. and Stanton, D. D., Transforming growth factor-beta enhances the ultraviolet-mediated stress response in p53−/−keratinocytes, Int. J. Oncol. 1998; 13(4):781-9) Chronic UVR exposure to the skin may lead to melanoma and squamous cell carcinomas of the skin. (Hildesheim J, Bulavin D V, Anver M R, et al., Gadd45a protects against UV irradiation-induced skin tumors, and promotes apoptosis and stress signaling via MAPK and p53, Cancer Res. 2002; 62(24):7305-15).
Severity of sunburn is related to duration of exposure, skin type and amount of protection. Potts J F, Sunlight, Sunburn, and Sunscreens, Postgrad. Med. 1990; 87:52-61. Factors influencing the cutaneous response to UVR depend on interactions among many other factors besides exposure time and dose. Wavelengths of the radiation source, skin characteristics such as pigmentation, hydration and skin thickness, and external factors such as wind, temperature and humidity all effect the response. Reflection off snow and sand may also lead to increased exposure. Some medications are known to be sensitizing to ultraviolet radiation. Tricyclic antidepressants, antihistamines, anti-infectives, antineoplastic agents, antipsychotic agents, diuretics, hypoglycemic agents, nonsteroidal anti-inflammatory drugs, and sunscreens all may decrease an individual's tolerance for sun exposure.
Current treatment for sunburn includes the systemic administration of aspirin or nonsteroidal anti-inflammatory drugs (NSAIDs) to inhibit the cyclooxygenase pathway and thereby reduce prostaglandin production. NSAIDs work best if administered within the first several hours after exposure. Systemic corticosteroids are often employed and probably shorten the course of the pain that accompanies severe sunburn. Corticosteroids should not be given to patients with severe, second-degree burns because they increase the risk of infection. Topical steroids show minimal, if any benefit.
Over-the-counter topical remedies include anesthetics such as lidocaine hydrochloride, benzocaine, and pramoxine hydrochloride. Skin soothing ingredients such as aloe vera, tocopheryl acetate (Vitamin E), menthol, camphor, eucalyptus oil, and calamine are also popular ingredients known in the art. Home topical remedies include taking a cool bath with oatmeal or baking soda; and spreading the juice of a cut potato, lavender essential oil, or chamomile on the burn.
While these topical remedies may help soothe the skin or temporarily relieve the pain associated with sunburn, they are not a treatment for the underlying inflammation that defines sunburn. Non-steroidal anti-inflammatories must be given systemically soon after exposure to be effective. Moreover, patients with allergies to NSAIDs, sensitive stomachs, or potential negative drug interactions may not be able to tolerate this treatment.
Current sunburn prevention methods include wearing protective clothing and avoidance of the sun during midday. But these methods restrict the outdoor activities, such as swimming, of a person wanting to avoid sunburn. Topical products for the prevention of sunburn fall into two categories: physical barriers and chemical absorbers. Chemical sunscreens are generally aromatic compounds conjugated with a carbonyl group. After application, the chemical sunscreen components diffuse into the stratum corneum and adsorb or conjugate with various proteins. Product effectiveness is determined by the depth of penetration, binding affinity for different proteins, and duration of protection. These chemicals absorb radiation in the UV spectrum. Chemical sunscreens have the disadvantages of possibly staining clothing and causing contact dermatitis. Moreover, recently concerns have been raised regarding the mutagenic properties of the most popular chemical sunscreens p-amino-benzoic acid (PABA) and PABA esters. Physical blockers, such as zinc oxide, talc, and titanium dioxide, reflect or scatter UVR. Many consumers find these products messy to apply and cosmetically unappealing.
Thus, a method for treating or preventing UV injury or sunburn is needed.
Scar Formation
Wounds caused by trauma or surgery are accompanied by an initial inflammatory response which is a natural response of the body and a first step of the wound healing process. The initial inflammatory response is followed by the formation of fibrous tissue, more commonly referred to as scar tissue, by proliferation of fibroblasts, and differentiation of fibroblasts to myofibroblasts, which produce collagen, mucopolysaccharides, and gylcosaminoglycans at the wound site. A certain amount of inflammation is required in the early healing stages in order to clear away the cellular and protein debris that accumulates at the wound to avoid infection and/or chronic inflammation. The second stage of wound healing involves a repair process which entails the influx and proliferation of fibrous tissue, due in part by the production of collagen and other substances by the fibroblasts, resulting in the formation of dense fibrous connective tissue that is visually seen as a scar.
The process of wound healing broadly comprises a regeneration phase and a repair phase, the differentiation between the two based on the resultant tissue. In regeneration, specialized tissues are replaced by the proliferation of surrounding undamaged specialized cells. In repair, lost tissue is replaced by granulation tissue which matures to form scar tissue. The repair phase involves the generation of the repair material, which for the majority of musculoskeletal injuries, involves the production of scar (collagen) material. Generation of repair material occurs fairly soon after injury, typically within 24-48 hours, and continues for a period of several weeks after injury, the time period depending in part on the amount of vasculature in the injured tissue. During this period, the bulk of the scar material is formed, with scar formation being evident and ultimately complete with a functional scar is achieved.
As mentioned above, inflammation is a normal and necessary prerequisite to healing. The inflammatory events involve both a vascular cascade of events and a cellular cascade of events. These occur in parallel and are significantly interlinked. The inflammatory cascade involves production of chemical mediators that make an active contribution to the healing process. For example, the cellular cascade involves emigration of neutrophils, monocytes, lymphocytes, eosinophils, basophils, to the wounded area and production of chemical mediators. The inflammatory response results in a vascular response, by production of a cellular and fluid exudate, with resulting edema. The course of the inflammatory response will depend upon the number of cells destroyed, the original causation of the process and the tissue condition at the time of insult.
Following the inflammation phase, the wound repair begins, with scar formation resulting. In some subjects, the scar tissue formation process results in what is referred to as hypertrophic or keloid scars. A keloid scar is a raised, firm, thickened red scar that exceeds the boundary of the injury and may grow for a prolonged period of time. A keloid scar occurs when the tissue response is out of proportion to the amount of scar tissue required for normal repair and healing. The increase in scar size is due to deposition of an increased amount of collagen into the tissue. Keloid development has been associated with different types of skin injury including surgery, ear piercing, laceration, burns, vaccination or inflammatory process. Common sites are earlobes and the upper trunk and extremities.
Scar formation is both a cosmetic problem and can in some cases be a medical problem. For example, scars on the face following an injury or surgery undesirable and can negatively impact a person. In some cases, keloid development occurs and a visible, undesirable scar results. Moreover, intra-abdominal adhesions results in a very significant morbidity and mortality in every surgery practice. Treatment of pelvic adhesions following surgery is often performed, and repeat surgery can greatly aggravate scarring.
There remains a need for a treatment to prevent scar formation, to reduce excessive scar formation and to prevent development of adhesions. Mechanical barriers are currently used to prevent adhesion formation, and these are only minimally effective clinically. Keloids have been treated with injection of corticosteroid into the scar, by laser therapy, and by administration of pharmacologic agents that interfere with collagen synthesis. Methods for improving the appearance of scars and for prevention excessive scarring and adhesions, without the inhibition of wound healing, are needed.
Measurement of Inhibition of c-Jun Terminal Kinase in Skin.
Previous studies have measured levels of IL-10, TNF-α, and NO in cells exposed to UVB irradiation either with or without specific inhibitors, demonstrating that UVB induced the production of those proinflammatory mediators, purportedly via activation of the p38 MAPK signalling pathway (Mutou Y, Tsukimoto M, Homma T, Kojima S, Immune Response Pathways in Human Keratinocyte (HaCat) Cells are Induced by Ultraviolet B via p38 Mitogen-activated Protein Kinase Activation, J. Health Science 2010; 56(6):675-83). Additionally, the induction of c-Jun protein and phosphorylation after UV irradiation of human skin has been reported (Fisher G, Talwar H, Lin J, Lin P, McPhillips F, Wang Z, Li X, Wan Y, Kang S, and Voorhees J, Retinoic Acid Inhibits Induction of c-Jun Protein by Ultraviolet Radiation That Occurs Subsequent To Activation Of Mitogen-Activated Protein Kinase Pathways In Human Skin In Vivo, J. Clin Invest. 1998; 101(6):1432-40 and Einspahr J, Bowden T, Alberts D, McKenzie N, Saboda K, Warneke J, Salasche S, Ranger-Moore J, Lewandrowski C, Nagle R, Nickoloff B, Brooks, C, Dong Z, and Stratton S, Cross-Validation of Murine UV Signal Transduction Pathways in Human Skin, Photochem. Photobiol. 2008; 84(2):463-76).
Clinical markers are needed to evaluate the biological effects of drug candidates. The methods set forth herein allow for the evaluation of the biological effects of JNK inhibitors and, accordingly, are useful in a clinical setting, such as by providing a straight-forward avenue for following the in vivo activity of a JNK inhibitor, and for assessing the sensitivity of a particular patient population to treatment with JNK inhibitors, in particular oral JNK inhibitors.
Citation or identification of any reference in Section 2 of this application is not to be construed as an admission that the reference is prior art to the present application.